It is often useful to determine the presence of particular substances within biological mixtures that may be separated by Liquid Chromatography. In many instances it is also desirable to quantify the amounts of these particular samples in the biological mixtures. Typically this would be done using a Triple Quadrupole mass spectrometer. Typically the mass spectrometer is set to multiple reaction monitoring mode (MRM). In MRM the mass spectrometer has its first quadrupole set to transmit the mass of only the desired parent ion. The collision cell then fragments the parent ions into numerous daughter ions, and the third quadrupole is set to transmit only a selected daughter ion produced by fragmentation of the parent ion. A detector measures the ion current that has been transmitted through the whole apparatus and produces a chromatogram for the specific fragment of the ion of interest. A threshold is set for the total ion current (TIC) such that when the total ion current exceeds the threshold the mass spectrometer switches modes from MRM to a confirmatory mode for a predetermined period of time at which the third quadrupole scans the through the mass range in order to obtain a spectrum of all the fragments produced. This spectrum acts as a confirmation that the parent ion is the ion anticipated to be the parent by review of the fragments produced.
The height of the chromatography peak and the area of the peak may both be used in order to calculate the quantity of the daughter ion that is present in the sample, and from this by comparison with a calibration curve, the quantity of the parent present.
However, switching from MRM to confirmatory mode limits the accuracy of the quantitation possible within the mass spectrometer. This is a particular problem when the Liquid Chromatograph is running at high pressure, which reduces the width of the chromatography peak. The triggering of the confirmatory scan will remove data from the chromatogram produced by the mass spectrometer. If the chromatography peak is narrow, this information may include the peak rather than solely information leading up to the peak, which has a severe impact on the capability of calculating the quantity of the daughter ions present because the data including the peak height being lost. This also prevents an accurate value for the peak area to be calculated.
It is therefore desired to produce a method of quantitation in a Liquid Chromatography/Mass spectrometry system which will be capable of measuring the peak height in all cases and also to allow a confirmation scan to be performed.